骨癌痛大鼠脊髓中WNK1激酶表达的变化
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摘要
目的研究骨癌痛大鼠脊髓中WNK1表达的变化。方法健康♀SD大鼠30只,体质量170 g~200 g,随机分为3组,对照组(C组,n=3)、假手术组(S组,n=3)和骨癌痛组(BCP组,n=24)。对照组不给予任何处理,假手术组、骨癌痛组分别在左胫骨上端注射PBS液5μL和WALKER 256乳腺癌细胞5μL(约1×10~5个细胞)。模型制备前d 1以及制备后d 3、6、9、10、11、12测定大鼠机械痛阈。C组与S组于术后d 12、BCP组于模型制备后d 3、6、9、12时处死大鼠并取脊髓(L4~6)。运用qRT-PCR及Western blot方法分别检测不同时间点WNK1 mRNA及蛋白表达。结果与S组比较,BCP组术后d 3机械痛阈开始降低(P<0.05),BCP组脊髓中WNK1 mRNA在术后d 3起表达上调并随时间推移呈递增趋势(P<0.01),至d 9达到峰值(P<0.05),随后表达下调,至d 12差异无统计学意义(P>0.05);WNK1蛋白在术后d 6开始表达上调,并随时间推移呈递增趋势至12 d(P<0.01)。结论骨癌痛大鼠脊髓中WNK1表达异常增高,可能参与了大鼠骨癌痛的发生和维持。
Aim To investigate the changes in the expression of WNK1 in spinal cord of a rat model with bone cancer pain. Methods Female SD rats,weighing 170 ~ 200 g,were randomly divided into three groups: normal control group( group C,n = 3),sham operation group( group S,n = 3) and bone cancer pain group( group BCP,n = 24). Group C was not given any treatment,and group S was injected into the bone marrow of left tibia with 5 μl PBS solution while group BCP with 5 μl WALKER 256 mammary gland cancer cell suspension( approximately 1 × 10~5 cells).Mechanical paw withdrawal threshold( MWT) was measured at d1 before inoculation( baseline) and d3,6,9,10,11,12 after inoculation. Group S and C were sacrificed at d 12 while group BCP at d 3,6,9,12 after inoculation and spinal cord( L4 ~ 6) were removed at different time points for detection of WNK1 mRNA expression by qRT-PCR and WNK1 protein expression by Western blot. Results Compared with group C and S,group BCP's MWT started to decrease since d 3( P< 0. 05). The mRNA expression of WNK1 in spinal cord was up-regulated( P < 0. 05) from d 3 showing an increasing trend over time until d 9( peak) and then down-regulated to d 12( P > 0. 05) while the protein expression upregulated since d6 and also showed an increasing trend to d 12( P < 0. 01). Conclusion The expression of WNK1 in spinal cord of a rat model with bone cancer pain increased abnormally,which may be involved in the occurrence and maintenance of a rat model with bone cancer pain.
Aim To investigate the changes in the expression of WNK1 in spinal cord of a rat model with bone cancer pain. Methods Female SD rats,weighing 170 ~ 200 g,were randomly divided into three groups: normal control group( group C,n = 3),sham operation group( group S,n = 3) and bone cancer pain group( group BCP,n = 24). Group C was not given any treatment,and group S was injected into the bone marrow of left tibia with 5 μl PBS solution while group BCP with 5 μl WALKER 256 mammary gland cancer cell suspension( approximately 1 × 10~5 cells).Mechanical paw withdrawal threshold( MWT) was measured at d1 before inoculation( baseline) and d3,6,9,10,11,12 after inoculation. Group S and C were sacrificed at d 12 while group BCP at d 3,6,9,12 after inoculation and spinal cord( L4 ~ 6) were removed at different time points for detection of WNK1 mRNA expression by qRT-PCR and WNK1 protein expression by Western blot. Results Compared with group C and S,group BCP's MWT started to decrease since d 3( P< 0. 05). The mRNA expression of WNK1 in spinal cord was up-regulated( P < 0. 05) from d 3 showing an increasing trend over time until d 9( peak) and then down-regulated to d 12( P > 0. 05) while the protein expression upregulated since d6 and also showed an increasing trend to d 12( P < 0. 01). Conclusion The expression of WNK1 in spinal cord of a rat model with bone cancer pain increased abnormally,which may be involved in the occurrence and maintenance of a rat model with bone cancer pain.
引文
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[9]Wang J,Zhang R,Dong C,et al.Topical treatment with Tong-LuoSan-Jie gel alleviates bone cancer pain in rats[J].J Ethnopharmacol,2012,143(3):905-13.
[10]Richardson C,Rafiqi F H,Karlsson H K,et al.Activation of the thiazide-sensitive Na+-Cl-cotransporter by the Wnk-regulated kinases spak and osr1[J].J Cell Sci,2008,121(5):675-84.
[11]Ahmed M M,Lee H,Clark Z,et al.Pathogenesis of spinal cord injury induced edema and neuropathic pain:Expression of multiple isoforms of Wnk1[J].Ann Neurosci,2014,21(3):97-103.
[12]Hasbargen T,Ahmed M M,Miranpuri G,et al.Role of Nkcc1 and Kcc2 in the development of chronic neuropathic pain following spinal cord injury[J].Ann N Y Acad Sci,1198(2010),168-72.
[13]He Y,Xu S,Huang J,et al.Analgesic effect of intrathecal bumetanide is accompanied by changes in spinal sodium-potassium-chloride co-transporter 1 and potassium-chloride co-transporter 2 expression in a rat model of incisional pain[J].Neural Regen Res,2014,9(10):1055-62.
[2]Lenertz L Y,Lee B H,Min X,et al.Properties of WNK1 and implications for other family members[J].J Biol Chem,2005,280(29):26653-8.
[3]Price T J,Cervero F,Gold M S,et al.Chloride regulation in the pain pathway[J].Brain Res Rev,2009,60(1):149-70.
[4]Price T J,Cervero F,de Koninck Y.Role of cation-chloride-cotransporters(CCC)in pain and hyperalgesia[J].Curr Top Med Chem,2005,5(6):547-55.
[5]何雁冰,徐世元,黄俊杰.鞘内注射布美他尼在大鼠足底切口痛模型中的镇痛作用[J].中国疼痛医学杂志,2014,7:463-6.[5]He Y B,Xu S Y,Huang J J.Analgesic effect of intrathecal injection of bumetanide in rats with postoperative incisional pain[J].Chin J Pain Med,2014,7:463-466.
[6]姚明,杨建平,王丽娜,等.腹水传代与体外培养Walker 256癌细胞系建立大鼠骨癌痛模型的可行性[J].中华医学杂志,2008,88(13):880-4.[6]Yao M,Yang J P,Wang L N,et al.Feasibility of establishment of rat model if bone cancer pain by using Walker 256 cells cultured in vitro or in vivo[J].Natl Med J China,2008,88(13):880-4.
[7]Brigatte P,Sampaio S C,Gutierrez V P,et al.Walker 256 tumorbearing rats as a model to study cancer pain[J].Pain,2007,8(5):412-21.
[8]Mao-Ying Q L,Wang X W,Yang C J,et al.Robust spinal neuroinflammation mediates mechanical allodynia in Walker 256 induced bone cancer rats[J].Mol Brain,2012,5:16.doi:10.1186/1756-6606-5-16.
[9]Wang J,Zhang R,Dong C,et al.Topical treatment with Tong-LuoSan-Jie gel alleviates bone cancer pain in rats[J].J Ethnopharmacol,2012,143(3):905-13.
[10]Richardson C,Rafiqi F H,Karlsson H K,et al.Activation of the thiazide-sensitive Na+-Cl-cotransporter by the Wnk-regulated kinases spak and osr1[J].J Cell Sci,2008,121(5):675-84.
[11]Ahmed M M,Lee H,Clark Z,et al.Pathogenesis of spinal cord injury induced edema and neuropathic pain:Expression of multiple isoforms of Wnk1[J].Ann Neurosci,2014,21(3):97-103.
[12]Hasbargen T,Ahmed M M,Miranpuri G,et al.Role of Nkcc1 and Kcc2 in the development of chronic neuropathic pain following spinal cord injury[J].Ann N Y Acad Sci,1198(2010),168-72.
[13]He Y,Xu S,Huang J,et al.Analgesic effect of intrathecal bumetanide is accompanied by changes in spinal sodium-potassium-chloride co-transporter 1 and potassium-chloride co-transporter 2 expression in a rat model of incisional pain[J].Neural Regen Res,2014,9(10):1055-62.